We plan to investigate the regulation of the scattered genes of the methionine regulon in E. coli by development of a system for the controlled expression of several of these genes in vitro, measurement of mRNA corresponding to these genes both in vivo and in vitro, and the conduct of appropriate in vivo experiments to verify and expand our in vitro results. We have available several different transducing phage carrying genes of the methionine regulon. Three of these phages carrying the metB metJ metF (and probably metLM) gene cluster have normal, controlled expression of the metB locus in vivo. These phages also have a functional metF locus as judged by growth of metF lysogens, and those derivatives carrying metJ ion alleles are able to repress methionine regulon genes in a metJ- host. We have synthesized radioactive substrates for the sensitive measurement of the metB and metC enzymes and have radioactive metF enzyme substrate. Using DNA from these and other transducing phages and S-30s from appropriate bacterial strains, we will measure in vitro synthesis of the met gene products (starting with metB). We will then investigate the control parameters in crude extracts and if the metJ gene product appears to be a regulator, we will proceed with its isolation and characterization. We will attempt to determine the level of control and to identify the regulatory effectors. If these experiments are successful, we will study the interaction of the metJ gene product with metDNA and the low molecular weight effectors. As necessary, we will isolate and characterize more transducing phage and will expand our studies to other loci. We have experience with current methods of transducing phage isolation but will investigate the feasibility of new methods using restriction enzymes. We also plan to investigate the genetics of the met regulon isolating many mutants of the loci we study, especially mutants of metJ to obtain altered gene products for studies to correlate structure and function.